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(S)-Adenosyl-L-methionine and the Enzymes That Use it: Inhibition, Mutagenesis and Kinetic Studies from the Biotin Biosynthesis and Methionine Salvage Pathways

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dc.contributor.advisor Jarrett, Joseph T.
dc.contributor.author Cramer, Julia Devine
dc.date.accessioned 2019-05-28T19:41:50Z
dc.date.available 2019-05-28T19:41:50Z
dc.date.issued 2018-12
dc.identifier.uri http://hdl.handle.net/10125/62255
dc.subject Biochemistry
dc.subject biotin biosynthesis
dc.subject enzyme inhibition
dc.subject enzyme kinetics
dc.subject kinetic isotope effects
dc.subject methionine salvage
dc.subject radical SAM
dc.title (S)-Adenosyl-L-methionine and the Enzymes That Use it: Inhibition, Mutagenesis and Kinetic Studies from the Biotin Biosynthesis and Methionine Salvage Pathways
dc.type Thesis
dc.contributor.department Chemistry
local.identifier.alturi http://dissertations.umi.com/hawii:10089
dcterms.abstract S-Adenosyl-L-methionine is a versatile cofactor utilized by many enzymes to catalyze diverse chemical reactions, including methylation, transamination, and radical initiation among others. This talk will discuss three enzymes, BioA, BioB, and MtnN. BioA and BioB use SAM to accomplish key steps in biotin biosynthesis. MtnN catalyzes the first step in metabolizing by-products of SAM-dependent reactions. De novo synthesis of biotin is critical for the survival of mycobacteria, including Mycobacterium tuberculosis, a human pathogen that causes the chronic respiratory condition of the same name. For this reason, enzymes in the biotin biosynthetic pathway have become one point of focus in the search for new antibiotics. Understanding the kinetics, mechanism, and substrate binding events for these enzymes is fundamental for the development of antimycobacterial agents. Mutagenesis studies in BioA reveal a dynamic enzyme, capable of retaining activity despite mutation of multiple residues, and improved activity upon point mutation of Phe-17. Analysis of the unusual kinetics of BioB turnover (marked by substrate inhibition and negative cooperativity) provides insight into subunit communication in this dimeric enzyme, and the role of quantum mechanical effects in radical enzyme catalysis. This talk will also feature studies on the inhibition of BioB by acidomycin, which has potent whole cell activity against extensively drug resistant tuberculosis. SAM-dependent enzymes form a variety of energy rich metabolites as by-products, including methylthioadenosine (MTA), S-adenosyl-homocysteine (SAH), and 5’-deoxyadenosine. The methionine salvage pathway exists in many organisms, and facilitates recycling of sulfur containing biomolecules. As recently as 2018, it was reported that E. coli are one of very few organisms that do not recycle MTA via this metabolic pathway. Evidence to the contrary and kinetic studies of the first enzyme in the pathway, MtnN will be presented. Comparison of MtnN turnover with four different substrates indicates the enzyme binds each with similar affinity, but reacts at varied rates.
dcterms.description Ph.D. Thesis. University of Hawaiʻi at Mānoa 2018.
dcterms.extent 115 pages
dcterms.language eng
dcterms.publisher University of Hawaiʻi at Mānoa
dcterms.rights All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
dcterms.type Text
Appears in Collections: Ph.D. - Chemistry


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